The structure of the IL-11 signalling complex provides insight into receptor variants associated with craniosynostosis.

Interleukin 11 (IL-11), a member of the IL-6 family of cytokines, has roles in haematopoiesis, inflammation, bone metabolism, and craniofacial development. IL-11 also has pathological roles in chronic inflammatory diseases, fibrosis, and cancer. In this structural snapshot, we explore our recently published cryo-EM structure of the human IL-11 signalling complex to understand the molecular mechanisms of complex formation and disease-associated mutations. IL-11 signals by binding to its cell surface receptors, the IL-11 receptor α subunit (IL-11Rα) and glycoprotein 130 (gp130), to form a hexameric signalling complex. We examine the locations within the complex of receptor sequence variants that are associated with craniosynostosis and craniosynostosis-like phenotypes and speculate on potential molecular mechanisms leading to defects in signalling function. While these causative amino acid sequence changes in IL-11Rα are generally distal to interfaces between components of the complex, important structural residues are highly represented, including proline residues, cysteine residues involved in disulfide bonds, and residues within or surrounding the tryptophan-arginine ladder. We also note the locations and potential effects of amino acid substitutions within the extracellular domains of gp130 that are associated with craniosynostosis. As focus on the physiological and pathological functions of IL-11 grows, the importance of high-resolution structural knowledge of IL-11 signalling to understand disease-associated mutations and to inform therapeutic strategies will only increase.

Unbiased Quantitative Proteomics of Organoid Models of Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) is a lethal solid malignancy with many patients succumbing to the disease within 6 months of diagnosis. The mechanisms that underlie PDAC initiation and progression are poorly understood. Current treatment options are primarily limited to chemotherapy, which is often provided with palliative intent. Unfortunately, there are no robust biomarkers to guide treatment selection or monitor treatment response. This is concerning given the increasing incidence of this cancer. We and others have generated organoid models to explore the biology underlying PDAC with the goal of identifying new therapeutic targets. Here we provide protocols to generate a preclinical PDAC organoid model and methods to use these to define the proteomic landscape of this cancer.

Identification of Serum Biomarkers to Monitor Therapeutic Response in Intestinal-Type Gastric Cancer.

There are a limited number of clinically useful serum biomarkers to predict tumor onset or treatment response in gastric cancer (GC). For this reason, we explored the serum proteome of the gp130Y757F murine model of intestinal-type gastric cancer (IGC). We identified 30 proteins with significantly elevated expression in early gp130Y757F IGC and 12 proteins that were significantly elevated in late gp130Y757F IGC compared to age- and gender-matched wild-type mice. Within these signatures, there was an overlap of 10 proteins commonly elevated in both early- and late-stage disease. These results highlight the potential to identify serum biomarkers of disease stage. Since IGC in the gp130Y757F model can be reversed following therapeutic inhibition of Interleukin (IL)-11, we explored whether the protein signatures we identified could be used to monitor tumor regression. We compared two different therapeutic modalities and found 5 proteins to be uniquely differentially expressed between control animals and animals halfway through treatment, with 10 differentially expressed at the end of treatment. Our findings highlight the potential to identify reliable biomarkers to track IGC tumor regression in response to treatment.

The E3 ubiquitin ligase NEDD4 regulates chemoresistance to 5-fluorouracil in colorectal cancer cells by altering JNK signalling.

Colorectal cancer (CRC) is the second leading cause of cancer deaths. Though chemotherapy is the main treatment option for advanced CRC, patients invariably acquire resistance to chemotherapeutic drugs and fail to respond to the therapy. Although understanding the mechanisms regulating chemoresistance has been a focus of intense research to manage this challenge, the pathways governing resistance to drugs are poorly understood. In this study, we provide evidence for the role of ubiquitin ligase NEDD4 in resistance developed against the most commonly used CRC chemotherapeutic drug 5-fluorouracil (5-FU). A marked reduction in NEDD4 protein abundance was observed in a panel of CRC cell lines and patient-derived xenograft samples that were resistant to 5-FU. Knockout of NEDD4 in CRC cells protected them from 5-FU-mediated apoptosis but not oxaliplatin or irinotecan. Furthermore, NEDD4 depletion in CRC cells reduced proliferation, colony-forming abilities and tumour growth in mice. Follow-up biochemical analysis highlighted the inhibition of the JNK signalling pathway in NEDD4-deficient cells. Treatment with the JNK activator hesperidin in NEDD4 knockout cells sensitised the CRC cells against 5-FU. Overall, we show that NEDD4 regulates cell proliferation, colony formation, tumour growth and 5-FU chemoresistance in CRC cells.

Structures of the interleukin 11 signalling complex reveal gp130 dynamics and the inhibitory mechanism of a cytokine variant.

Interleukin (IL-)11, an IL-6 family cytokine, has pivotal roles in autoimmune diseases, fibrotic complications, and solid cancers. Despite intense therapeutic targeting efforts, structural understanding of IL-11 signalling and mechanistic insights into current inhibitors are lacking. Here we present cryo-EM and crystal structures of the human IL-11 signalling complex, including the complex containing the complete extracellular domains of the shared IL-6 family ß-receptor, gp130. We show that complex formation requires conformational reorganisation of IL-11 and that the membrane-proximal domains of gp130 are dynamic. We demonstrate that the cytokine mutant, IL-11 Mutein, competitively inhibits signalling in human cell lines. Structural shifts in IL-11 Mutein underlie inhibition by altering cytokine binding interactions at all three receptor-engaging sites and abrogating the final gp130 binding step. Our results reveal the structural basis of IL-11 signalling, define the molecular mechanisms of an inhibitor, and advance understanding of gp130-containing receptor complexes, with potential applications in therapeutic development.

Confocal Endomicroscopy Monitoring of Tumor Formation.

The utilization of preclinical murine models of colorectal cancer (CRC) has been essential to our understanding of the onset and progression of disease. As the genetic complexity of these models evolves to better recapitulate emerging CRC subtypes, our ability to utilize these models to discover and validate novel therapeutic targets will also improve. This will be aided, in part, by the development of live animal imaging techniques, including confocal endomicroscopy for mice. Here in this chapter, we describe the combined use of standard white light endoscopy and confocal endomicroscopy thereby providing a method to rapidly image and assess changes in the colon of an individual live mouse in real time. These methods permit the generation of high-resolution cross-sectional images of the tumor microenvironment for immediate visualization of cells of interest, avoiding the need for euthanasia and tissue collection across multiple cohorts of mice.

The Interleukin-11/IL-11 Receptor Promotes Glioblastoma Survival and Invasion under Glucose-Starved Conditions through Enhanced Glutaminolysis.

Glioblastoma cells adapt to changes in glucose availability through metabolic plasticity allowing for cell survival and continued progression in low-glucose concentrations. However, the regulatory cytokine networks that govern the ability to survive in glucose-starved conditions are not fully defined. In the present study, we define a critical role for the IL-11/IL-11Rα signalling axis in glioblastoma survival, proliferation and invasion when cells are starved of glucose. We identified enhanced IL-11/IL-11Rα expression correlated with reduced overall survival in glioblastoma patients. Glioblastoma cell lines over-expressing IL-11Rα displayed greater survival, proliferation, migration and invasion in glucose-free conditions compared to their low-IL-11Rα-expressing counterparts, while knockdown of IL-11Rα reversed these pro-tumorigenic characteristics. In addition, these IL-11Rα-over-expressing cells displayed enhanced glutamine oxidation and glutamate production compared to their low-IL-11Rα-expressing counterparts, while knockdown of IL-11Rα or the pharmacological inhibition of several members of the glutaminolysis pathway resulted in reduced survival (enhanced apoptosis) and reduced migration and invasion. Furthermore, IL-11Rα expression in glioblastoma patient samples correlated with enhanced gene expression of the glutaminolysis pathway genes GLUD1, GSS and c-Myc. Overall, our study identified that the IL-11/IL-11Rα pathway promotes glioblastoma cell survival and enhances cell migration and invasion in environments of glucose starvation via glutaminolysis.

S100 family proteins are linked to organoid morphology and EMT in pancreatic cancer.

Epithelial-mesenchymal transition (EMT) is a continuum that includes epithelial, partial EMT, and mesenchymal states, each of which is associated with cancer progression, invasive capabilities, and ultimately, metastasis. We used a lineage-traced sporadic model of pancreatic cancer to generate a murine organoid biobank from primary and secondary tumors, including sublines that underwent partial EMT and complete EMT. Using an unbiased proteomics approach, we found that organoid morphology predicts the EMT state, and the solid organoids are associated with a partial EMT signature. We also observed that exogenous TGFß1 induces solid organoid morphology that is associated with changes in the S100 family, complete EMT, and the formation of high-grade tumors. S100A4 may be a useful biomarker for predicting EMT state, disease progression, and outcome in patients with pancreatic cancer.

Fecal DNA Virome Is Associated with the Development of Colorectal Neoplasia in a Murine Model of Colorectal Cancer.

Alteration of the gut virome has been associated with colorectal cancer (CRC); however, when and how the alteration takes place has not been studied. Here, we employ a longitudinal study in mice to characterize the gut virome alteration in azoxymethane (AOM)-induced colorectal neoplasia and identify important viruses associated with tumor growth. The number and size of the tumors increased as the mice aged in the AOM treated group, as compared to the control group. Tumors were first observed in the AOM group at week 12. We observed a significantly lower alpha diversity and shift in viral profile when tumors first appeared. In addition, we identified novel viruses from the genera Brunovirus, Hpunavirus that are positively associated with tumor growth and enriched at a late time point in AOM group, whereas members from Lubbockvirus show a negative correlation with tumor growth. Moreover, network analysis revealed two clusters of viruses in the AOM virome, a group that is positively correlated with tumor growth and another that is negatively correlated with tumor growth, all of which are bacteriophages. Our findings suggest that the gut virome changes along with tumor formation and provides strong evidence of a potential role for bacteriophage in the development of colorectal neoplasia.

Combined Treatment with a WNT Inhibitor and the NSAID Sulindac Reduces Colon Adenoma Burden in Mice with Truncated APC.

Adenomatous polyposis coli (APC) truncations occur in many colorectal cancers and are often associated with immune infiltration. The aim of this study was to determine whether a combination of Wnt inhibition with anti-inflammatory (sulindac) and/or proapototic (ABT263) drugs can reduce colon adenomas. Apc min/+ and doublecortin-like kinase 1 (Dclk1)Cre/+ ;Apc fl/fl mice were exposed to dextran sulphate sodium (DSS) in their drinking water to promote the formation of colon adenomas. Mice were then treated with either a Wnt-signaling antagonist pyrvinium pamoate (PP), an anti-inflammatory agent sulindac or proapoptotic compound ABT263 or a combination of PP+ABT263, or PP+sulindac. Colon adenoma frequency, size, and T-cell abundance were measured. DSS treatment resulted in significant increases in colon adenoma number (P < 0.001, n > 5) and burden in Apc min/+ (P < 0.01, n > 5) and Dclk1 Cre/+ ;Apc fl/fl (P < 0.02, n > 5) mice. There was no effect on adenomas following treatment with PP in combination with ABT263. Adenoma number and burden were reduced with PP+sulindac treatment in Dclk1 Cre/+;Apc fl/fl mice (P < 0.01, n > 17) and in Apc min/+ mice (P < 0.001, n > 7) treated with sulindac or PP+sulindac with no detectable toxicity. PP treatment of Apc min/+ mice increased the frequency of CD3+ cells in the adenomas. The combination of Wnt pathway inhibition with sulindac was more effective in Dclk1 Cre/+;Apc fl/fl mice and provides an opportunity for killing Apc-mutant colon adenoma cells, indicating a strategy for both colorectal cancer prevention and potential new treatments for patients with advanced colorectal cancer. Outcomes from the results of this study may be translatable to the clinic for management of FAP and other patients with a high risk of developing colorectal cancer. Significance: Colorectal cancer is one of the most common cancers worldwide with limited therapeutic options. APC and other Wnt signaling mutations occur in the majority of colorectal cancers but there are currently no Wnt inhibitors in the clinic. The combination of Wnt pathway inhibition with sulindac provides an opportunity for killing Apc-mutant colon adenoma cells and suggests a strategy for colorectal cancer prevention and new treatments for patients with advanced colorectal cancer.

Emerging roles for IL-11 in inflammatory diseases.

Interleukin-11 (IL-11) is a cytokine that has been strongly implicated in the pathogenesis of fibrotic diseases and solid malignancies. Elevated IL-11 expression is also associated with several non-malignant inflammatory diseases where its function remains less well-characterized. Here, we summarize current literature surrounding the contribution of IL-11 to the pathogenesis of autoimmune inflammatory diseases, including rheumatoid arthritis, multiple sclerosis, diabetes and systemic sclerosis, as well as other chronic inflammatory conditions such as periodontitis, asthma, chronic obstructive pulmonary disease, psoriasis and colitis.

The Diverse Applications of Pancreatic Ductal Adenocarcinoma Organoids.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal solid malignancies. While immortalized cancer cell lines and genetically engineered murine models have increased our understanding of PDAC tumorigenesis, they do not recapitulate inter- and intra-patient heterogeneity. PDAC patient derived organoid (PDO) biobanks have overcome this hurdle, and provide an opportunity for the high throughput screening of potential new therapies. This review provides a summary of the PDAC PDO biobanks established to date, and discusses how they have advanced our understanding of PDAC biology. Looking forward, the development of coculturing techniques for specific immune or stromal cell populations will enable a better understanding of the crosstalk that occurs within the tumor microenvironment, and the impact of this crosstalk on treatment response.

Functional and structural analysis of cytokine-selective IL6ST defects that cause recessive hyper-IgE syndrome.

BACKGROUND: Biallelic variants in IL6ST, encoding GP130, cause a recessive form of hyper-IgE syndrome (HIES) characterized by high IgE level, eosinophilia, defective acute phase response, susceptibility to bacterial infections, and skeletal abnormalities due to cytokine-selective loss of function in GP130, with defective IL-6 and IL-11 and variable oncostatin M (OSM) and IL-27 levels but sparing leukemia inhibitory factor (LIF) signaling. OBJECTIVE: Our aim was to understand the functional and structural impact of recessive HIES-associated IL6ST variants. METHODS: We investigated a patient with HIES by using exome, genome, and RNA sequencing. Functional assays assessed IL-6, IL-11, IL-27, OSM, LIF, CT-1, CLC, and CNTF signaling. Molecular dynamics simulations and structural modeling of GP130 cytokine receptor complexes were performed. RESULTS: We identified a patient with compound heterozygous novel missense variants in IL6ST (p.Ala517Pro and the exon-skipping null variant p.Gly484_Pro518delinsArg). The p.Ala517Pro variant resulted in a more profound IL-6- and IL-11-dominated signaling defect than did the previously identified recessive HIES IL6ST variants p.Asn404Tyr and p.Pro498Leu. Molecular dynamics simulations suggested that the p.Ala517Pro and p.Asn404Tyr variants result in increased flexibility of the extracellular membrane-proximal domains of GP130. We propose a structural model that explains the cytokine selectivity of pathogenic IL6ST variants that result in recessive HIES. The variants destabilized the conformation of the hexameric cytokine receptor complexes, whereas the trimeric LIF-GP130-LIFR complex remained stable through an additional membrane-proximal interaction. Deletion of this membrane-proximal interaction site in GP130 consequently caused additional defective LIF signaling and Stüve-Wiedemann syndrome. CONCLUSION: Our data provide a structural basis to understand clinical phenotypes in patients with IL6ST variants.

Type 2 Innate Lymphoid Cells Protect against Colorectal Cancer Progression and Predict Improved Patient Survival.

Chronic inflammation of the gastrointestinal (GI) tract contributes to colorectal cancer (CRC) progression. While the role of adaptive T cells in CRC is now well established, the role of innate immune cells, specifically innate lymphoid cells (ILCs), is not well understood. To define the role of ILCs in CRC we employed complementary heterotopic and chemically-induced CRC mouse models. We discovered that ILCs were abundant in CRC tumours and contributed to anti-tumour immunity. We focused on ILC2 and showed that ILC2-deficient mice developed a higher tumour burden compared with littermate wild-type controls. We generated an ILC2 gene signature and using machine learning models revealed that CRC patients with a high intratumor ILC2 gene signature had a favourable clinical prognosis. Collectively, our results highlight a critical role for ILC2 in CRC, suggesting a potential new avenue to improve clinical outcomes through ILC2-agonist based therapeutic approaches.

Necroptosis is dispensable for the development of inflammation-associated or sporadic colon cancer in mice.

Chronic inflammation of the large intestine is associated with an increased risk of developing colorectal cancer (CRC), the second most common cause of cancer-related deaths worldwide. Necroptosis has emerged as a form of lytic programmed cell death that, distinct from apoptosis, triggers an inflammatory response. Dysregulation of necroptosis has been linked to multiple chronic inflammatory diseases, including inflammatory bowel disease and cancer. Here, we used murine models of acute colitis, colitis-associated CRC, sporadic CRC, and spontaneous intestinal tumorigenesis to investigate the role of necroptosis in these gastrointestinal pathologies. In the Dextran Sodium Sulfate-induced acute colitis model, in some experiments, mice lacking the terminal necroptosis effector protein, MLKL, or its activator RIPK3, exhibited greater weight loss compared to wild-type mice, consistent with some earlier reports. However, the magnitude of weight loss and accompanying inflammatory pathology upon Mlkl deletion varied substantially between independent repeats. Such variation provides a possible explanation for conflicting literature reports. Furthermore, contrary to earlier reports, we observed that genetic deletion of MLKL had no impact on colon cancer development using several mouse models. Collectively, these data do not support an obligate role for necroptosis in inflammation or cancer within the gastrointestinal tract.

A Biobank of Colorectal Cancer Patient-Derived Xenografts.

Colorectal cancer (CRC) is a challenging disease, with a high mortality rate and limited effective treatment options, particularly for late-stage disease. Patient-derived xenografts (PDXs) have emerged as an informative, renewable experimental resource to model CRC architecture and biology. Here, we describe the generation of a biobank of CRC PDXs from stage I to stage IV patients. We demonstrate that PDXs within our biobank recapitulate the histopathological and mutation features of the original patient tumor. In addition, we demonstrate the utility of this resource in pre-clinical chemotherapy and targeted treatment studies, highlighting the translational potential of PDX models in the identification of new therapies that will improve the overall survival of CRC patients.

Emerging roles for the IL-6 family of cytokines in pancreatic cancer.

Pancreatic cancer has one of the poorest prognoses of all malignancies, with little improvement in clinical outcome over the past 40 years. Pancreatic ductal adenocarcinoma is responsible for the vast majority of pancreatic cancer cases, and is characterised by the presence of a dense stroma that impacts therapeutic efficacy and drives pro-tumorigenic programs. More specifically, the inflammatory nature of the tumour microenvironment is thought to underlie the loss of anti-tumour immunity and development of resistance to current treatments. Inflammatory pathways are largely mediated by the expression of, and signalling through, cytokines, chemokines, and other cellular messengers. In recent years, there has been much attention focused on dual targeting of cancer cells and the tumour microenvironment. Here we review our current understanding of the role of IL-6, and the broader IL-6 cytokine family, in pancreatic cancer, including their contribution to pancreatic inflammation and various roles in pancreatic cancer pathogenesis. We also summarise potential opportunities for therapeutic targeting of these pathways as an avenue towards combating poor patient outcomes.

Structural Understanding of Interleukin 6 Family Cytokine Signaling and Targeted Therapies: Focus on Interleukin 11.

Cytokines are small signaling proteins that have central roles in inflammation and cell survival. In the half-century since the discovery of the first cytokines, the interferons, over fifty cytokines have been identified. Amongst these is interleukin (IL)-6, the first and prototypical member of the IL-6 family of cytokines, nearly all of which utilize the common signaling receptor, gp130. In the last decade, there have been numerous advances in our understanding of the structural mechanisms of IL-6 family signaling, particularly for IL-6 itself. However, our understanding of the detailed structural mechanisms underlying signaling by most IL-6 family members remains limited. With the emergence of new roles for IL-6 family cytokines in disease and, in particular, roles of IL-11 in cardiovascular disease, lung disease, and cancer, there is an emerging need to develop therapeutics that can progress to clinical use. Here we outline our current knowledge of the structural mechanism of signaling by the IL-6 family of cytokines. We discuss how this knowledge allows us to understand the mechanism of action of currently available inhibitors targeting IL-6 family cytokine signaling, and most importantly how it allows for improved opportunities to pharmacologically disrupt cytokine signaling. We focus specifically on the need to develop and understand inhibitors that disrupt IL-11 signaling.